The present invention relates to vectors containing a small DNA segment called a "restriction site bank" which comprises a large number of unique cloning sites and which permits the cloning of any foreign DNA material into prokaryotic and eukaryotic cells. The invention also relates to processes for the construction of restriction site banks and to the use of vectors containing such restriction site banks.
It is well known that restriction enzymes play a key role in recombinant DNA technology, e.g., in the generation of complementary cohesive ends for a cloning vector and for the DNA to be cloned, thus allowing for the recombination of the vector and DNA. A large number of such restriction enzymes which recognize specific, well-defined sites are now available, providing great versatility in cloning experiments, but only when the corresponding sites are present in the vector of choice. Moreover, these sites should be present in limited number and should preferably be unique, so as to reduce as much as possible the number of recombination events necessary to obtain the desired recombinant DNA.
Previous development of plasmid vectors for cloning into prokaryotes has concentrated on the use of multiple antibiotic resistance genes containing unique restriction sites, so that cloning at one of these sites results in sensitivity to an antibiotic (insertional inactivation), thereby facilitating detection of recombinants. While extremely useful, this approach has its limitations. First, it is difficult to construct a plasmid containing a large number of unique restriction sites since addition of other antibiotics resistance genes introduces duplicates sites and at the same time leads to an undesirable increase in plasmid size. Second, there are many situations in which insertional inactiviation is not useful, e.g., when screening for specific rare recombinants in shotgun experiments, or when a suitable unique cloning site in an antibiotic resistance gene is not available. In the case of eukaryotes, the use of plasmid vectors with multiple antibiotic resistance genes is of little interest, as eukaryotic cells are generally resistant to most antibiotics.
Because of these limitations, techniques have been developed which allow screening for hybrid colonies in other ways, e.g., by using hybridization probes, by complementation of genetically distinguishable host strains, or by simply screening a large number of plasmid DNA preparations using restriction enzymes. Similarly, the proportion of nonrecombinant, parental-type plasmids can be reduced by alkaline phosphatase treatment of the vector prior to ligation, or by the cloning of fragments between two different noncomplemetary restriction sites.